Mechanisms within the secretory and endocytic pathways exist to ensure that each organelle in eukaryotic cells obtain its correct allotment of resident proteins. Defects in proper maintenance of specific organellar protein transport are the basis for many human diseases such as cancer and Alzheimer's syndrome. Two integral approaches, genetics and inadequate understanding remains in transfer of proteins from the pre- lysosomal compartment (PLC) to the lysosome. Resident lysosomal proteins pass through the PLC after being sorted away from secretory proteins in the trans Golgi network. Similarly, proteins destined for degradation after endocytosis from the cell surface pass through the PLC (or late endosome) before their turnover in the lysosome. With these multiple protein traffic directions, eukaryotic cells must combined many events for proper sorting, targeting, and delivery of proteins from the PLC. The greatest source of confusion about the PLC concerns whether carrier vesicles transport material to lysosomes or if the PLC undergoes a maturation process, changing into a lysosome. Studies that combine genetic, molecular, and biochemical approaches will lead to the complete understanding of this intercompartmental protein transport problem. The yeast, Saccharomyces cerevisiae, contains not only a lysosome-like vacuole, but also a PLC-like prevacuolar compartment (PVC). This proposal describes the use of cell-free reconstitution assays in vitro, yeast mutant analyses in vivo, and morphological techniques to clarify transport mechanisms between the PVC and the vacuole. These facile approaches have produced evidence to support the notion that specific carrier vesicles act to transport protein cargo from the PVC to the vacuole. Moreover, recent data suggests that these organelles may not be single entities in yeast. This putative complexity would give the yeast vacuolar system even more similarity to the lysosomal system of mammalian cells. The following specific aims are proposed to prove or disprove these hypotheses. (1) Elucidate the function of cytosolic proteins involved in delivery of resident hydrolases to the vacuole/lysosome via the biogenesis and function of the prevacuolar compartment. (3) Purify and characterize membrane-bound compartments that comprise organelles of the post- Golgi/vacuolar system in yeast to discover novel marker proteins. These organelles include prevacuolar compartments, PVC-derived intermediate vesicles or subcompartments, and vacuoles.